Abstract

Superoxide anion is produced during normal cellular respiration and plays key roles in cellular physiology with its dysregulation being associated with a variety of diseases. Superoxide anion is a short-lived molecule and, therefore, its homeostatic regulation and role in biology and disease requires dynamic quantification with fine temporal resolution. Here we validated coelenterazine as a reporter of intracellular superoxide anion concentration and used it as a dynamic measure both in vitro and in vivo. Chemiluminescence was dependent upon superoxide anion levels, including those produced during cellular respiration, and concentrations varied both kinetically and temporally in response to physiologically relevant fluctuations in glucose levels. In vivo imaging with coelenterazine revealed that beta cells of the pancreas have increased levels of superoxide anion, which acted as a measure of beta-cell function and mass and could predict the susceptibility of mice to diabetes mellitus. Glucose response and regulation are key elements of cellular physiology and organismal biology, and superoxide anion appears to play a fundamental and dynamic role in both of these processes.

Superoxide anion concentration, as detected with coelenterazine, serves as an in vivo reporter of beta-cell mass.

(A) Images of a representative nonprogressive mouse at 10, 12, 14 and 16 weeks of age. (B) Images of a representative progressive mouse at 10, 12, 14 and 16 weeks of age. The progressive mouse was normoglycemic at the time of the images. (C) The signal intensities for the two groups of mice, nonprogressive (white) (n = 8) and progressive (black) (n = 7), for a given age normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. (D) The signal intensities for the nonprogressive mice during the last week of the study normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. The signal intensities for progressive mice for the last day of the study during which their fasting blood glucose levels were not exceeding 5.6, 6.9 or 8.3 mmol/L normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. (E) The mean of the fasting blood glucose levels that were less than 8.3 mmol/L for nonprogressive and progressive mice. (Error bars represent the mean ± SEM, and * denotes a significant difference between the groups of P < 0.039.)